Pharmaceutical composition having site-specific delivery

ABSTRACT

Use of α-hydroxy acids and poly-α-hydroxy acids as spacer between a therapeutically and/or diagnostically active compound and a soluble macromolecular carrier in pharmaceutical compositions having site-specific delivery. In one embodiment glycolic acid, L-lactic acid or tetra-L-lactic acid is used as spacer between a non-steroidal anti-inflammatory substance and a carrier of low molecular protein (LMWP).

This application is a 371 of PCT/NL/93/00061, filed Mar. 15, 1993.

The invention relates to a pharmaceutical composition havingsite-specific and in particular tissue-specific delivery, in addition toa method for producing same.

Pharmaceutical compositions having site-specific delivery are sometimesmade by starting from an inactive variant ("prodrug") of atherapeutically or diagnostically active substance which is convertedinto the active form only after reaching a determined place in the bodysuch as a specific organ or tissue. Another possibility is the combiningof the active substance with a pharmaceutical carrier in particle form,such as liposomes for instance, or a soluble macromolecular carrier,such as polypeptides for instance, which have a preference for aspecific place in the human or animal body and there release the activesubstance.

In the case that a polypeptide or other soluble macromolecular materialis used as carrier for the active substance, it is preferred to couplethis carrier with the active substance by covalent bonds. On arrival atthe desired location in the body these covalent bonds then have to bebroken, this such that the active substance is released. Problems canhowever occur here. Despite the fact that many macromolecular carriersare biologically degradable, they sometimes do not release the activesubstance in the correct form or at the desired rate, In order toobviate this problem molecules of a compound serving as spacer can belinked between the active substance and the carrier, but the selectionof a suitable spacer results in turn in new problems. With a view to anefficient and/or controlled decoupling of the active substance and thecarrier, particular attention must be given to the nature of the activesubstance, the type of covalent bond of the active substance to thespacer and also to the length and branching degree of the spacer. Thespacer itself and its breakdown products must further be non-toxic.

It has now been found during further research that α-hydroxy acids andpoly-α-hydroxy acids are eminently suitable for use as spacer between anactive substance and a soluble macromolecular carrier provided theactive substance has a terminal carboxyl group. The α-hydroxy acids cannamely be bonded by esterification (between the α-hydroxy group of theα-hydroxy acid and the carboxyl group of the active substance) to theactive substance and be moreover coupled by any covalent bond (betweenthe carboxyl group of the α-hydroxy acid and a reactive group of themacromolecular carrier) to the soluble macromolecular carrier. Bothtypes of bonds are normally resistant to the conditions in thebloodstream of a human or animal body but, after arrival at a tissue atwhich the macromolecular carrier is specifically targeted, the esterbond between active substance and spacer could easily be broken byenzymes (esterases), so that the active substance is released in theoriginal (active) form. Since the α-hydroxy acids and poly-α-hydroxyacids are not toxic and allow of relatively easy coupling anddecoupling, they represent an attractive option for the selection of aspacer in pharmaceutical compositions of the stated type. It hasmoreover been found that the rate of delivery of the active substanceinto the desired tissues can be controlled by variation of the type ofα-hydroxy acid and also by variation of the length and/or the branchingdegree of the poly-α-hydroxy acid that is used as spacer in thepharmaceutical composition.

The invention therefore provides a pharmaceutical composition havingsite-specific delivery and comprising:

at least one therapeutically and/or diagnostically active compound, saidcompound having a terminal carboxyl group,

a soluble macromolecular pharmaceutical carrier, and

an α-hydroxy acid or poly-α-hydroxy acid functioning as a spacer betweenactive compound and carrier and being coupled through an ester bond tothe active compound and through any covalent bond to the carrier withthe exception of the conjugate of dextran and naproxen linked via aglycolic acid spacer. This conjugate was already disclosed in an articleof Larsen, C. in International Journal of Pharmaceutics 51, 233-240(1989).

The spacer for use in the composition according to the invention is anα-hydroxy acid or poly-α-hydroxy acid. Suitable examples are monobasicα-hydroxy acids such as glycolic acid and lactic acid as well as dibasicand tribasic α-hydroxy acids such as malic acid, citramalic acid,tartaric acid and citric acid. Understood by poly-α-hydroxy acids arecompounds which are formed by linking together (mutual esterification)of two or more molecules of α-hydroxy acid; a suitable example istetra-L-lactic acid which consists of four lactic acid units linkedtogether.

Any therapeutically and/or diagnostically active compound which has aterminal carboxyl group can be coupled by means of the α-hydroxy acidsor poly-α-hydroxy acids to a macromolecular pharmaceutical carrier.Suitable for use are for instance the substances known as "non-steroidalanti-inflammatory drugs" (NSAID), with as suitable examplesacetylsalicylic acid, (S) -6-methoxy-α-methyl-2-naphthalene acetic acidand the like.

Any soluble macromolecular pharmaceutical carrier can be coupled throughthe α-hydroxy acids and poly-α-hydroxy acids to a therapeutically and/ordiagnostically active compound. These are generally proteins,glycoproteins, polypeptides and polyclonal or monoclonal antibodieswhich can each display a selective targeting to a specific tissue typeor a specific type of tissue call. Monoclonal antibodies are for exampletargeted especially at tissues with a specific type of antigen, whileglycoproteins with terminal sugar residues are particularly targeted atspecific types of liver cell. Good results are achieved with a group ofpeptides known as Low Molecular Weight Proteins (LMWP) with as suitableexamples lysozyme, cytochrome C and aprotein. These LMWPs are targetedspecially at the kidneys. They are rapidly cleared out of thebloodstream by glomerular filtration and then quantitatively reabsorbedin the proximal tubular cells, whereafter they are broken down to aminoacids by the lysosomes.

Good results were obtained with a pharmaceutical composition wherein thetherapeutically active substance (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid was bonded via an α-hydroxy acid to lysozyme. It was foundhere that the composition remained stable in the bloodstream ofexperimental animals but was cleaved in the kidneys, wherein thetherapeutically active substance was released in active form. It wasalso found here that the ester bonds in combinations with L-lactic acidare cleaved more rapidly than in combinations with glycolic acid andthat chain lengthening of the spacer makes the ester bonds still betteraccessible for cleaving by enzymes. This indicates that all α-hydroxyacids and poly-α-hydroxy acids are usable as spacer and also creates thepossibility of achieving a controlled delivery of the active substanceinto the tissues by variation of the type of α-hydroxy acid and byvariation of the length and/or branching degree of the α-hydroxy acid orpoly-α-hydroxy acid.

The compositions according to the invention can in general be producedby coupling an α-hydroxy acid or poly-α-hydroxy acid throughesterification of the α-hydroxy group to a therapeutically ordiagnostically active compound having a terminal carboxyl group on oneside and coupling with its free carboxyl group through a covalent bondto a soluble macromolecular carrier on the other side. Both reactionscan be performed in any manner usual for this purpose. Theesterification can for instance be performed by allowing an acidchloride or other reactive derivative of the active compound to reactdirectly with the α-hydroxy acid or poly-α-hydroxy acid. Anotherpossibility is to apply one of the usual methods of peptide chemistrysuch as an esterification under the influence ofdicyclohexylearbodiimide. It is desirable in that case to initiallyprotect the carboxyl group of the α-hydroxy acid or poly-α-hydroxy acidby arranging a protective group and after esterification to remove thisprotective group in the usual manner, for example with trifluoroaceticacid and anisole.

When the macromolecular carrier consists of a polypeptide the carboxylgroup of the α-hydroxy acid or poly-α-hydroxy acid can be coupled to theamino group of a terminal amino acid in this polypeptide. Such acoupling can take place in a manner usual in peptide chemistry, forinstance with a carbodiimide method or an N-hydroxysuccinimide orN-hydroxysulphosuccinimide method. Should the carrier consist of aprotein, glycoprotein or of antibodies, similar procedures can then befollowed.

The obtained coupling products can be purified in usual manner. For thepurpose of the pharmaceutical application they can be complemented withusual excipients, diluents and additives. The composition obtained willgenerally take the form of an injection composition, but other dosageforms are not excluded. The dosage to be used will conform to the activesubstance incorporated in the composition.

There now follow a number of preparation examples and biological tests.The term "naproxen" refers to (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid.

EXAMPLE I

Naproxen-L-lactic acid-lysozyme

1) L-lactic acid-PMB. A suspension of L-lactic acid (1.5 g, 10 mmol) indimethylformamide was treated with triethylamine (20 mmol) andpentamethylbenzyl chloride (PMBC1) (10 mmol). The mixture was heatedcarefully to obtain a solution and held at room temperature at night.Thereafter an excess of 1N NaHCO, was added. Within several minutes theester separated out in crystalline form. The product was collected,washed with water and dried under vacuum.

Yield 95%. Melting point 115°-116° C. ¹ H NMR (CDCl₃): δ 5.27 (m, 2,CH₂), 4.23 (q, 1, CHCH₃), 2.27 (s, 15, CH₃ --Cq), 1.47 (d, 3, CH₃ CH).

2) Naproxen-L-lactic acid-PMB. Added to a solution of naproxen (2.3 g,10 mmol), L-lactic acid-PMB (2.5 g, 10 mmol) and4-dimethylamino-pyridine (1.22 g, 10 mmol) in 150 ml dichloromethane wasa solution of dicyclohexylcarbodiimide (2.27 g, 11 mmol) in 50 mldichloromethane. The reaction mixture was stirred at 25° C., wherein theprogress of the reaction was followed with thin-layer chromatography.Thereafter the N,N-dicyclohexylurea was filtered off. The filtrate waswashed with 1M KHSO₄ (2×20 ml), water (2×20 ml), and 5% NaHCO₃ (2×20ml). The organic layer was dried above water-free sodium sulphate andevaporated dry in vacuo. The residue was washed with petroleum ether andheld under high vacuum for many hours to obtain an analytically pureproduct.

Yield 70%. ¹ H NMR (CDCl₃): δ 7.69-7.12 (m, 6, aromatic), 5.27 (m, 2,CH₂), 5.10 (q, 1, CHCH₃ (lact)), 3.94 (q, 1, CHCH₃ (naproxen)), 3.93 (s,3, CH₃ O), 2.27 (s, 15, CH₃ --Cq), 1.61 (d, 3, CH₃ CH (naproxen)), 1.47(d, 3, CH₃ CH (lact)).

3) Naproxen-L-lactic acid. A mixture of naproxen-L-lactic acid-PMB (2.3g, 5 mmol), anisole (12 ml) and trifluoroacetic acid (10 ml) was held atroom temperature for 2 minutes. The excess of reagent was then removedunder vacuum below 30° C. The residue was dissolved in dichloromethane(100 ml) and washed with water (4×20 ml). The organic layer wasextracted with diethylether (2×50 ml). Acidifying with 6N HCl providedthe product which was extracted with dichloromethane (4×25 ml). Thewashed and dried product (Na₂ SO₄) was evaporated dry and the residuedried in vacuo at 50° C. The product was crystallized fromdichloromethane/cyclohexane.

Yield 75%. ¹ H NMR (CDCl₃): δ 1055-1050 (br s, 1, OH), 7.54-6.93 (m, 6,aromatic), 4.97 (q, 1, CHCH₃ (lact)), 3.76 (q, 1, CHCH₃ (naproxen)),3.69 (s, 1, CH₃ O), ₁.40 (d, 5, CH₃ OH (naproxen)), 1.31 (d, 3, CH₃ CH(lact)).

4) Naproxen-L-lactic acid-NHS. Naproxen-L-lactic acid (302 mg, 1 mmol)was dissolved in 10 ml dimethylformamide. Dicyclohexylcarbodiiaide (277mg, 1.1 mmol) was then added. The solution was stirred for 15 minutes,whereafter N-hydroxysuccinimide (115 mg, 1 mmol) dried beforehand invacuo for 24 hours at 50° C. was added. The mixture was stirred for 24hours. After filtering off the precipitation the filtrate was evaporateddry in vacuo and the residue was washed with dry heptane. The residuewas dissolved in ethylacetate, filtered, evaporated dry in vacuo andcrystallized from dichloromethane/hexane.

Yield 91%. 1H NMR (CDCl₃): δ 7.5-6.9 (m, 6, aromatic), 5.0 (q, 1, CHCH₃(lact)), 3.8 (q,1, CHCH₃), 3.7 (s, 3, CH₃ O), 2.8 (s, 4, CH₂ CH₂ (NHS)),₁.5 (d, 3, CH₃ CH (naproxen)), 1.3 (d, 3, CH₃ CH (lact)).

5) Naproxen-L-lactic acid-lysozyme. Naproxen-L-lactic acid-NHS (14.1 mg,34.7 μmol) was dissolved in 10 ml DMF and placed in reaction for 2 hourswith lysozyme (100 mg, 6.95 μmol) in a DMF/borate (0.025M; pH 8.5)(20/80) mixture. After filtration of the precipitated material thefiltrate was purified by gel filtration. After a subsequentultrafiltration (Amicon) and lyophylisation, the product was kept at-20° C. Yield 74%. The molar substitution degree was 0.6 as determinedwith a fluorimetric measurement of naproxen (excitation wavelength 330nm, emission wavelength 360 nm) and a protein test according to Bradford(compare Bradford, Anal. Biochem. 72, 248 (1976)).

EXAMPLE II

Naproxen-ester derivatives.

In small-scale tests the acid chloride of naproxen (10.8 mg, 0.04 mmol)was dissolved together with glycolic acid (4 Mg, 0.04 mmol), L-lacticacid (5 mg, 0.04 mmol) or tetra-L-lactic acid (12.6 mg, 0.04 =mol) indry dichloromethane. Triethylamine (11 microliters, 0.08 mmol) wasadded, whereafter the mixture was stirred for 18 hours. The progress ofthe reaction was followed with thin-layer chromatography. The obtainedester derivatives were purified by HPLC with reverse phase.

Several biological tests were carried out with the thus obtainedproducts.

Test 1

During in vitro tests the naproxen esters obtained in Example II wereincubated at diverse pH values with lysosomelyzates obtained fromhomogenates of rat kidneys. At pH 5 it was found that 81% of thenaproxen was released from the ester with glycolic acid within 24 hours.In contrast, 100% naproxen had already been released from an eater withL-lactic acid within 30 minutes. In the case of the ester of naproxenand tetra-L-lactic acid the ester bond was found to be still moresensitive to enzymatic cleaving in vitro.

Test 2

During in vivo tests male Wistar rats (280-310 g) were placed inmetabolic cages where they had free access to food and water. Afteraddition of 500 IU of heparin, 10 mg or 1 mg naproxen-L-lacticacid-lysozyme, freshly dissolved in blood plasma, was administered tothe rats by intravenous injection. Plasma samples and urine samples werecollected at regular intervals and analyzed. It was found that theinjected products were sufficiently stable in blood plasma to reach thekidneys intact. It was further found that the whole dose was reabsorbedinto the kidneys and locally metabolized to naproxen.

We claim:
 1. A conjugate adapted for site-specific delivery of an activecompound, said compound having an activity selected from the groupconsisting of therapeutic activity and diagnostic activity and having aterminal carboxyl group, wherein the active compound is linked via anester bond to a spacer that is covalently bonded to a solubleproteinaceous macromolecular pharmaceutical carrier, the spacer being amonomer or polymer of an α-hydroxy acid, said α-hydroxy acid beingselected from the group consisting of glycolic acid, L-lactic acid,malic acid, citramalic acid, tartaric acid and citric acid and whereinsaid conjugate is absorbable by a cell.
 2. A conjugate as claimed inclaim 1, wherein the spacer is tetra-L-lactic acid.
 3. A conjugate asclaimed in claim 1, wherein the active compound is a non-steroidalanti-inflammatory drug.
 4. A conjugate as claimed in claim 3, whereinthe active compound is (S)-naproxen.
 5. A conjugate as claimed in claim1, wherein the carrier is selected from the group consisting ofglycoproteins, polypeptides and polyclonal or monoclonal antibodies. 6.A conjugate as claimed in claim 5, wherein the carrier is a lowmolecular weight protein.
 7. A conjugate as claimed in claim 6, whereinthe carrier is a lysozyme.
 8. A method of producing a conjugate asclaimed in claim 1, which comprises providing the spacer, said spacerhaving an α-hydroxy group and a free carboxyl group; coupling the spacerthrough esterification of the α-hydroxy group to the active compound;and covalently bonding the spacer with the free carboxyl group to themacromolecular carrier.
 9. A conjugate according to claim 1, whereinsaid α-hydroxy acid is selected from the group consisting of glycolicacid and L-lactic acid.
 10. A conjugate according to claim 9, whereinthe active compound is a non-steroidal anti-inflammatory drug.
 11. Aconjugate according to claim 9, wherein the carrier is selected from thegroup consisting of glycoproteins, polypeptides and polyclonal ormonoclonal antibodies.
 12. A conjugate according to claim 9, wherein thecarrier is a low molecular weight protein.
 13. A conjugate according toclaim 12, wherein the carrier is a lysozyme.
 14. A method of producingthe conjugate according to claim 10, said method comprising providingthe spacer, said spacer having an α-hydroxy group and a free carboxylgroup; coupling the spacer through esterification of the α-hydroxy groupto the active compound; and covalently bonding the spacer with the freecarboxyl group to the carrier.
 15. A pharmaceutical compositioncomprising the conjugate according to claim 1 and an excipient.
 16. Acomposition according to claim 15, wherein said composition is suitablefor injection.
 17. A composition according to claim 16, wherein saidinjection is intravenous.
 18. A method of diagnosing or treating acondition responsive to said active compound in a patient comprisingadministering to said patient a composition according to claim
 15. 19. Amethod according to claim 18, wherein said administering is byintravenous injection.